When the innermost layer of the cornea, the endothelium, is damaged, for example from trauma (e.g., from cataract surgery), disease or dystrophy, the cornea swells with fluid (edema) and loses its optical clarity. Patients consequently suffer from vision loss and pain, and their only option to treat advanced disease is with corneal transplant surgery (also known as penetrating keratoplasty, PK) or Descemet's stripping endothelial keratoplasty (DSAEK), both technically difficult procedures that are very invasive to the patient and have significant limitations, such as the number of donor corneas available.
Recent studies have proposed the use of human corneal endothelial cells (HCECs) obtained from cadaveric donors to replace the damaged cells. See, e.g., Joyce and Zhu, Cornea. 2004 November; 23(8 Suppl):S8-S19; Engelmann, et al., Exper. Eye Res., vol. 78, no. 3, pp. 573-578, 2004. A potential advantage to such an approach could be the expansion of HCECs ex vivo before implantation into patients, thereby overcoming the limited tissue availability. HCECs can be expanded in defined tissue culture media for at least 5 passages, greatly expanding the number of cells derived from a single donor.
One of the main problems with such a technique is that the lack of defined surface markers specific for HCECs makes it difficult to confirm the identity of HCECs after several passages, or to select HCECs away from contaminating cells, or to identify the subset of HCECs that are likely to have the highest clinical efficacy from among the full population of HCECs, as current identification criteria are limited to cell morphology and the expression of functional genes, such as ATP1A1 (see, e.g., Kaye and Tice. Invest Ophthalmol. 1966; 522-32: Leuenberger and Novikoff, J Cell Biol. 1974: 60721-731; McCartney et al. Curr Eye 1987; 61479-1486) or the tight junction marker zonula occludens-1 (ZO-1) (see, e.g., Petroll et al., Curr Eye Res. 1999 January; 18(1):10-9), neither of which are specific to HCECs. It is also difficult to isolate HCECs from contaminant fibroblasts in culture, from neighboring cells in whole corneas, or from residual corneas from DSAEK.
In this regard, the current isolation method for obtaining HCECs from intact corneas comprises a peel-off step, where the endothelium and its basement membrane (Descemet's membrane) are peeled off the stroma and collected. See, e.g., Ko-Hua Chen et al., “Transplantation of Adult Human Corneal Endothelium Ex Vivo: A Morphologic Study,” Cornea 20(7): 731-737, 2001. The tissue collected thus contains HCECs, but it may also contain corneal keratocytes (specialized fibroblasts residing the stroma). Corneal keratocytes (also referred to herein simply as “keratocytes”) are undesirable contaminants in the HCECs culture, as they grow faster than the latter cells and they can take over the culture dish, thus making the final product essentially useless. In addition to residual stromal tissue, keratocytes may also arise from human endothelial cells which transform spontaneously into other types of cells such as keratocytes (see, e.g., G S. L. Peh et al., “Optimization of Human Corneal Endothelial Cells for Culture: The Removal of Corneal Stromal Fibroblast Contamination Using Magnetic Cell Separation,” International Journal of Biomaterials, Volume 2012 (2012), Article ID 601302, 8 pages.)